This application is a 371 of PCT/GB91/01578, filed Sep. 16, 1991.
The present invention relates to altered antibodies and their preparation. The invention is typically applicable to the production of humanised antibodies.
Antibodies typically comprise two heavy chains linked together by disulphide bonds and two light chains. Each light chain is linked to a respective heavy chain by disulphide bonds. Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end. The light chain variable domain is aligned with the variable domain of the heavy chain. The light chain constant domain is aligned with the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.
The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
The preparation of an altered antibody in which the CDRs are derived from a different species than the framework of the antibody""s variable domains is disclosed in EP-A-0239400. The CDRs may be derived from a rat or mouse monoclonal antibody. The framework of the variable domains, and the constant domains, of the altered antibody may be derived from a human antibody. Such a humanised antibody elicits a negligible immune response when administered to a human compared to the immune response mounted by a human against a rat or mouse antibody. Humanised CAMPATH-1 antibody is disclosed in EP-A-0328404.
We have now devised a new way of preparing an altered antibody. In contrast to previous proposals, this involves altering the framework of a variable domain rather than the CDRs. This approach has the advantages that it does not require a pre-existing cDNA encoding, for example, a human framework to which to reshape and that it is technically easier than prior methodologies.
Accordingly, the present invention provides a process for the preparation of an antibody chain in which the CDRs of the variable domain of the antibody chain are derived from a first mammalian species and the framework of the variable domain and, if present, the or each constant domain of the antibody chain are derived from a second different mammalian species, which process comprises:
(i) mutating the framework-encoding regions of DNA encoding a variable domain of an antibody chain of the said first species such that the mutated framework-encoding regions encode the said framework derived from the said second species; and
(ii) expressing the said antibody chain utilising the mutated DNA from step (i).
A variable domain of either or both chains of an antibody can therefore be altered by:
(a) determining the nucleotide and predicted amino acid sequence of a variable,domain of a selected antibody chain of the said first species;
(b) determining the antibody framework to which the framework of the said variable domain is to be altered;
(c) mutating the framework-encoding regions of DNA encoding the said variable domain such that the mutated framework-encoding regions encode the framework determined upon in step (b);
(d) linking the mutated DNA obtained in step (c) to DNA encoding a constant domain of the said second species and cloning the DNA into an expression vector; and
(e) introducing the expression vector into a compatible host cell and culturing the host cell under such conditions that antibody chain is expressed.
The antibody chain may be co-expressed with a complementary antibody chain. At least the framework of the variable domain and the or each constant domain of the complementary chain generally are derived from the said second species also. A light chain and a heavy chain may be co-expressed. Either or both chains may have been prepared by the process of the invention. Preferably the CDRs of both chains are.derived from the same selected antibody. An antibody comprising both expressed chains can be recovered.
The antibody preferably has the structure of a natural antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fabxe2x80x2)2 fragment, a Fab fragment, a light chain dimer or a heavy chain. The antibody may be an IgG such as an IgG1, IgG2, IgG3 or IgG4 IgM, IgA, IgE or IgD. Alternatively, the antibody may be a chimaeric antibody of the type described in WO 86/01533.
A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically, the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobulin region is fused at its C-terminus to the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence.
The invention is preferably employed to humanise an antibody, typically a monoclonal antibody and, for example, a rat or mouse antibody. The framework and constant domains of the resulting antibody are therefore human framework and constant domains whilst the CDRs of the light and/or heavy chain of the antibody are rat or mouse CDRs. Preferably all CDRs are rat or mouse CDRs. The antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat or mouse CDRs.
The process of the invention is carried out in such a way that the resulting antibody retains the antigen binding capability of the antibody from which it is derived. An antibody is reshaped according to the invention by mutating the framework-encoding regions of DNA coding for the variable domains of the antibody. This antibody and the reshaped antibody should both be capable of binding to the same antigen.
The starting antibody is typically an antibody of a selected specificity. In order to ensure that this specificity is retained, the variable domain framework of the antibody is preferably reshaped to about the closest variable domain framework of an antibody of another species. By xe2x80x9cabout the closestxe2x80x9d is meant about the most homologous in terms of amino acid sequences. Preferably there is a homology of at least 50% between the two variable domains.
There are four general steps to reshape a monoclonal antibody. These are:
(1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy chain variable domains;
(2) designing the reshaped antibody, i.e. deciding which antibody framework region to use during the reshaping process;
(3) the actual reshaping methodologies/techniques; and
(4) the transfection and expression of the reshaped antibody.
These four steps are explained below in the context of humanising an antibody. However, they may equally well be applied when reshaping to an antibody of a non-human species.
Step 1: Determining the Nucleotide and Predicted Amino Acid Sequence of the Antibody Light and Heavy Chain Variable Domains
To reshape an antibody only the amino acid sequence of antibody""s heavy and light chain variable domains needs to be known. The sequence of the constant domains is irrelevant because these do not contribute to the reshaping strategy. The simplest method of determining an antibody""s variable domain amino acid sequence is from cloned cDNA encoding the heavy and light chain variable domain.
There are two general methods for cloning a given antibody""s heavy and light chain variable domain cDNAs: (1) via a conventional cDNA library, or (2) via the polymerase chain reaction (PCR). Both of these methods are widely known. Given the nucleotide sequence of the cDNAs, it is a simple matter to translate this information into the predicted amino acid sequence of the antibody variable domains.
Step 2: Designing the Reshaped Antibody
There are several factors to consider in deciding which human antibody sequence to use during the reshaping. The reshaping of light and heavy chains are considered independently of one another, but the reasoning is basically similar for each.
This selection process is based on the following rationale: A given antibody""s antigen specificity and affinity is primarily determined by the amino acid sequence of the variable region CDRs. Variable domain framework residues have little or no direct contribution. The primary function of the framework regions is to hold the CDRs in their proper spacial orientation to recognize antigen. Thus the substitution of rodent CDRs into a human variable domain framework is most likely to result in retention of their correct spacial orientation if the human variable domain is highly homologous to the rodent variable domain from which they originated. A human variable domain should preferably be chosen therefore that is highly homologous to the rodent variable domain(s).
A suitable human antibody variable domain sequence can be selected as follows:
1. Using a computer program, search all available protein (and DNA) databases for those human antibody variable domain sequences that are most homologous to the rodent antibody variable domains. This can be easily accomplished with a program called FASTA but other suitable programs are available. The output of a suitable program is a list of sequences most homologous to the rodent antibody, the percent homology to each sequence, and an alignment of each sequence to the rodent sequence. This is done independently for both the heavy and light chain variable domain sequences. The above analyses are more easily accomplished if customized sub-databases are first created that only include human immunoglobulin sequences. This has two benefits. First, the actual computational time is greatly reduced because analyses are restricted to only those sequences of interest rather than all the sequences in the databases. The second benefit is that, by restricting analyses to only human immunoglobulin sequences, the output will not be cluttered by the presence of rodent immunoglobulin sequences. There are far more rodent immunoglobulin sequences in databases than there are human.
2. List the human antibody variable domain sequences that have the most overall homology to the rodent antibody variable domain (from above). Do not make a distinction between homology within the framework regions and CDRs. Consider the overall homology.
3. Eliminate from consideration those human sequences that have CDRs that are a different length than those of the rodent CDRs. This rule-does not apply to CDR 3, because the length of this CDR is normally quite variable. Also, there are sometimes no or very few human sequences that have the same CDR lengths as that of the rodent antibody. If this is the case, this rule can be loosened, and human sequences with one or more differences in CDR length can be allowed.
4. From the remaining human variable domains, the one is selected that is most homologous to that of the rodent.
5. The actual reshaped antibody (the end result) should contain CDRs derived from the rodent antibody and a variable domain framework from the human antibody chosen above.
Step 3: The Actual Reshaping Methodologies/techniques
A cDNA encoding the desired reshaped antibody is preferably made beginning with the rodent cDNA from which the rodent antibody variable domain sequence(s) was originally determined. The rodent variable domain amino acid sequence is compared to that of the chosen human antibody variable domain sequence. The residues in the rodent variable domain framework are marked that need to be changed to the corresponding residue in the human to make the rodent framework identical to that of the human framework. There may also be residues that need adding to or deleting from the rodent framework sequence to make it identical to that of the human.
Oligonucleotides are synthesised that can be used to mutagenize the rodent variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.
The advantages of this method of reshaping as opposed to splicing CDRs into a human framework are that (1) this method does not require a pre-existing cDNA encoding the human framework to which to reshape and (2) splicing CDRs is technically more difficult because there is usually a large region of poor homology between the mutagenic oligonucleotide and the human antibody variable domain. This is not so much a problem with the method of splicing human framework residues onto a rodent variable domain because there is no need for a pre-existing cDNA encoding the human variable domain. The method starts instead with the rodent cDNA sequence. Also, splicing framework regions is technically easier because there is a high degree of homology between the mutagenic oligonucleotide and the rodent variable domain framework. This is true because a human antibody variable domain framework has been selected that is most homologous to that of the rodent.
The advantage of the present method of reshaping as opposed to synthesizing the entire reshaped version from scratch is that it is technically easier. Synthesizing a reshaped variable domain from scratch requires several more oligonucleotides, several days more work, and technical difficulties are more likely to arise.
Step 4: The Transfection and Expression of the Reshaped Antibody
Following the mutagenesis reactions to reshape the antibody, the cDNAs are linked to the appropriate DNA encoding light or heavy chain constant region, cloned into an expression vector, and transfected into mammalian cells. These steps can be carried out in routine fashion. A reshaped antibody may therefore be prepared by a process comprising:
a) preparing a first replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least a variable domain of an Ig heavy or light chain, the variable domain comprising framework regions from a first antibody and CDRs comprising at least parts of the CDRs from a second antibody of different specificity;
b) if necessary, preparing a second replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least the variable domain of a complementary Ig light or heavy chain respectively;
c) transforming a cell line with the first or both prepared vectors; and
d) culturing said transformed cell line to produce said altered antibody.
Preferably the DNA sequence in step a) encodes both the variable domain and the or each constant domain of the antibody chain, the or each constant domain being derived from the first antibody. The antibody can be recovered and purified. The cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus. Most preferably, the immortalised cell line is a myeloma cell line or a derivative thereof.
Although the cell line used to produce the altered antibody is preferably a mammalian cell line, any other suitable cell line, such as a bacterial cell line or a yeast cell line, may alternatively be used. In particular, it is envisaged that E. colixe2x80x94derived bacterial strains could be used.
It is known that some immortalised lymphoid cell lines, such as myeloma cell lines, in their normal state secrete isolated Ig light or heavy chains. If such a cell line is transformed with the vector prepared in step (a) it will not be necessary to carry out step (b) of the process, provided that the normally secreted chain is complementary to the variable domain of the Ig chain encoded by the vector prepared in step (a).
However, where the immortalised cell line does not secrete or does not secrete a complementary chain, it will be necessary to carry out step (b). This step may be carried out by further manipulating the vector produced in step (a) so that this vector encodes not only the variable domain of an altered antibody light or heavy chain, but also the complementary variable domain.
Alternatively, step (b) is carried out by preparing a second vector which is used to transform the immortalised cell line. This alternative leads to easier construct preparation, but may be less preferred than the first alternative in that it may not lead to as efficient production of antibody.
In the case where the immortalised cell line secretes a complementary light or heavy chain, the transformed cell line may be produced for example by transforming a suitable bacterial cell with the vector and then fusing the bacterial cell with the immortalised cell Line by spheroplast fusion. Alternatively, the DNA may be directly introduced into the immortalised cell line by electroporation or other suitable method.
An antibody is consequently produced in which CDRs of a variable domain of an antibody chain are homologous with the corresponding CDRs of an antibody of a first mammalian species and in which the framework of the variable domain and the constant domains of the antibody are homologous with the corresponding framework and constant domains of an antibody of a second, different, mammalian species. Typically, all three CDRs of the variable domain of a light or heavy chain are derived from the first species.
The present process has been applied to obtain an antibody against human CD4 antigen. Accordingly, the invention also provides an antibody which is capable of binding to human CD4 antigen, in which the CDRs of the light chain of the antibody have the amino acid sequences:
CDR1: LASEDIYSDLA (SEQ ID NO:13)
CDR2: NTDTLQN (SEQ ID NO:14)
CDR3: QQYNNYPWT (SEQ ID NO:15),
in which the CDRs of the heavy chain of the antibody have the amino acid sequences:
CDR1: NYGMA (SEQ ID NO:16)
CDR2: TISHDGSDTYFRDSVKG (SEQ ID NO:17)
CDR3: QGTIAGIRH (SEQ ID NO:18), and
in which the framework of the variable domain and, if present, the or each constant domain of each chain are derived from a mammalian non-rat species.
The antibody preferably has the structure of a natural antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fabxe2x80x2)2 fragment, a Fab fragment, a light chain dimer or a heavy chain.
The antibody may be an IgG such as IgG1, IgG2, IgG3 or IgG4 IgM, IgA, IgE or IgD. Alternatively, the antibody may be a chimaeric antibody of the type described in WO 86/01533.
A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobulin region is fused at its C-terminus to the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence.
The invention is preferably employed to humanise a CD4 antibody such as a rat or mouse CD4 antibody. The framework and the constant domains of the resulting antibody are therefore human framework and constant domains whilst the CDRs of the light and/or heavy chain of the antibody are rat or mouse CDRs. Preferably all CDRs are rat or mouse CDRs. The antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat or mouse CDRs.
Preferably the framework of the antibody heavy chain is homologous to the corresponding framework of the human antibody KOL (Schmidt et al, Hoppe-Seyler""s Z. Physiol. Chem., 364 713-747, 1983). The sixth residue of framework 4 in this case is suitably Thr or Pro, preferably Thr. This residue is the 121st residue in the KOL antibody heavy chain variable region (Schmidt et al, 1983), and is identified as residue 108 by Kabat (Kabat et al, xe2x80x9cSequences of proteins of immunological interestxe2x80x9d, US Dept of Health and Human Services, US Government Printing Office, 1987). Alternatively, the framework of the antibody heavy chain is homologous to the corresponding framework of the human antibody NEW (Saul et al, J. Biol. Chem. 2: 585-597, 1978). The final residue of framework 1 in this case is suitably Ser or Thr, preferably Ser. This residue is at position 30 (Kabat et al, 1987). Preferably the framework of the antibody light chain is homologous to the variable domain framework of the protein REI (Epp et al, Eur. J. Biochem., 45, 513-524, 1974).
The framework regions of one or both chains of a CD4 antibody can be reshaped by the present process. Alternatively, one or both chains of a CD4 antibody may be reshaped by the procedure described in EP-A-0239400. The procedure of EP-A-0239400 involves replacing CDRs rather than the replacement of frameworks. The CDRs are grafted onto a framework derived from a mammalian non-rat species, typically a human. This may be achieved by oligonucleotide-directed in vitro mutagenesis of the CDR-encoding regions of an antibody chain, light or heavy, from a mammalian non-rat species. The oligonucleotides in such an instance are selected so that the resulting CDR-grafted antibody has the light chain CDRs 1 to 3 and the heavy chain CDRs 1 to 3 shown above.
The reshaped CD4 antibody can be used to induce tolerance to an antigen. It can be used to alleviate autoimmune diseases such as rheumatoid arthritis. It can be used to prevent graft rejection. Tolerance to a graft such as an organ graft or a bone marrow transplantation can be achieved. Also, the reshaped CD4 antibody might be used to alleviate allergies. Tolerance to allergens could be achieved.
The CD4 antibody may be depleting or non-depleting. A depleting antibody is an antibody which depletes more than 50%, for example from 90 to 99%, of target cells in vivo. A non-depleting antibody depletes fewer than 50%, for example, from 10 to 25% and preferably less than 10% of target cells in vivo. A CD4 antibody may be administered alone or may be co-administered with a non-depleting or depleting CD8 antibody. The CD4 antibody, depleting or non-depleting, and CD8 monoclonal antibody, depleting or non-depleting, may be administered sequentially in any order or may be administered simultaneously. An additional antibody, drug or protein may be administered before, during or after administration of the antibodies.
A CD4 antibody and, indeed, a CD8 antibody as appropriate are given parenterally, for example intravenously. The antibody may be administered by injection or by infusion. For this purpose the antibody is formulated in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent. Any appropriate carrier or diluent may be employed, for example phosphate-buffered saline solution.
The amount of non-depleting or depleting CD4 and, if desired, CD8 antibody administered to a patient depends upon a variety of factors including the age and weight of a patient, the condition which is being treated and the antigen(s) to which it is desired to induce tolerance. In a model mouse system from 1 xcexcg to 2 mg, preferably from 400 xcexcg to 1 mg, of a mAb is administered at any one time. In humans from 3 to 500 mg, for example from 5 to 200 mg, of antibody may be administered at any one time. Many such doses may be given over a period of several weeks, typically 3 weeks.
A foreign antigen(s) to which it is desired to induce tolerance can be administered to a host before, during, or after a course of CD4 antibody (depleting or non-depleting) and/or CDs antibody (depleting or non-depleting). Typically, however, the antigen(s) is administered one week after commencement of antibody administration, and is terminated three weeks before the last antibody administration.
Tolerance can therefore be induced to an antigen in a host by administering non-depleting or depleting CD4 and CD8 mAbs and, under cover of the mAbs, the antigen. A patient may be operated on surgically under cover of the non-depleting or depleting CD4 and CD8 mAbs to be given a tissue transplant such as an organ graft or a bone marrow transplant. Also, tolerance may be induced to an antigen already possessed by a subject. Long term specific tolerance can be induced to a self antigen or antigens in order to treat autoimmune disease such as multiple sclerosis or rheumatoid arthritis. The condition of a patient suffering from autoimmune disease can therefore be alleviated.